SIGNIFICANCE: Our long-term goal is to discover novel regulatory components of a core mechanism of cellular communication, i.e., signaling via heterotrimeric G proteins (henceforth trimeric G proteins), and to characterize the molecular mechanisms that underlie their involvement in human disease. Our emphasis is on cytoplasmic factors that regulate G protein signaling, which have been (and remain) understudied compared to G protein-coupled receptors (GPCRs), the main activating inputs for trimeric G proteins. During the last grant cycle, we made significant advances in this area by achieving all the originally proposed goals, thereby establishing a new paradigm of G protein regulation. We demonstrated the existence of a family of cytoplasmic activators of G proteins, dissected the structural basis for their molecular activity, and established the consequences of their dysregulation in specific cellular processes and human diseases. The specific goal of this renewal application is to characterize the prototype member of a potential new class of G protein regulators that has been linked to chronic pain and epilepsy. The achievement of our goals would provide deep mechanistic insights into a new paradigm of GPCR-G protein regulation that fine tunes inhibitory neuromodulation, and establish a new framework to devise therapeutic strategies for neurological disorders like chronic pain and epilepsy. BACKGROUND: In the course of a screen of proteins that bind to G?i subunits of G?-G?? timeric complexes, we identified a protein that regulates G proteins via a unique and novel mechanism. We have coined the term ?paradoxical G protein regulator? (PGR) to convey that it upregulates the modulation of some G protein effectors while simultaneously downregulating the modulation of other G protein effectors. Loss of this ?PGR? is known to alter GPCR signaling in neurons of the peripheral nervous system and causes chronic pain. It has also been linked to epilepsy. Despite its clear biomedical importance, the molecular mechanism by which this G protein regulator operates, and how it modulates neurotransmission in the brain are completely unknown. SYNOPSIS OF AIMS: Based on compelling preliminary data, we propose that the PGR modulates both G?i- and G??-dependent signaling without directly affecting the G protein enzymatic activity (i.e., nucleotide binding and/or hydrolysis), and that this novel mechanism fine tunes GPCR signaling in brain neurons to influence seizure susceptibility. In Aim#1 we will dissect how the PGR regulates G proteins at the molecular level by characterizing how it engages physically G?i and the consequences of this physical engagement on G protein signaling to different effectors. In Aim#2 we will characterize how it regulates GPCR signaling and neurotransmission at the cellular level by using primary cultures of neurons and brain slices from wild-type and KO mice. In Aim#3 we will determine the PGR's role at the physiological level by establishing how it impacts seizure susceptibility using genetically modified mouse models.